This research program investigates immune responses induced by viral and plasmid vectors. Results are relevant to vaccine potency and mechanisms of action. In addition, immune responses can alter the safety and efficacy of gene therapy using viral or plasmid vectors, especially in the context of readministration, either by blocking therapy or by causing immunopathology. Better understanding of such immune responses can help select predictors of clinical success as well as adverse events, and thus contribute to improved regulatory decision-making. Our work focuses on the mouse influenza system, and especially on heterosubtypic immunity: Animals immunized against influenza A virus have cross-protection against challenge with flu A of different subtypes. Vaccine development and prevention of pandemics would be aided by a more complete understanding of the broad cross-protection against widely divergent viral strains that is observed in animals. Main projects: a) Role of IgA: The role of local, mucosal immune responses in protection against influenza, and in particular in cross-protection against virus strains differing from the immunizing strain, is not well understood. We have performed mucosal immunizations and heterosubtypic challenges in knockout mice lacking IgA, to analyze the role of this specialized mucosal antibody. Results showed these mice were capable of responses providing cross-protection of both the upper and lower respiratory tract. b) Cross-protection in Ig-/- mice: Many vaccines based on T cell immunity (HIV, hepatitis, flu) have been proposed when antibodies are ineffective or too narrow in viral strain specificity, but the protective efficacy of T cells acting in the absence of antibody has not been tested in most cases. Using mice lacking all antibodies due to disruption of Ig H and L chain genes, we have previously shown protection against influenza B challenge, and have demonstrated that optimal protection requires both CD4+ and CD8+ T cells. We have now extended those studies to the influenza A virus system. Results of challenge studies showed that immunization with live virus conferred protection against heterosubtypic challenge. Thus T cells acting alone can mediate broad cross-protection. In vivo depletion demonstrated that both CD4+ and CD8+ T cells were required during the challenge period for optimal protection. c) Cross-protection in mice lacking certain double negative T cells: We have studied knockout mice lacking # T cells, and shown them capable of heterosubtyic immunity protective against challenge. In addition, knockout mice lacking NKT cells restricted by the non-classical MHC antigen CD1 were protected in heterosubtypic challenge experiments. d) Immune responses induced by plasmid DNA: DNA vaccines encoding conserved influenza virus antigens reduce challenge virus replication and the resulting morbidity and mortality. We had shown last year that vaccination with DNA encoding influenza nucleoprotein and matrix (NP+M) antigens protected animals, and that either CD4+ or CD8+ T cells could function without the other subset to protect the animal. We are currently investigating mechanisms by which CD4+ cells protect in the absence of CD8+ cells. In other recent work, we have shown that 8 months after NP+M DNA vaccination, mice are still protected against lethal challenge. Additional studies show that protection against lethal challenge is conferred by M DNA without DNA encoding NP, the major CTL target antigen, previously thought to be key. d) Potential pandemic subtypes: One advantage of DNA vaccination with conserved components is the broad range of viral variants against which it protects. We are studying the effect of DNA vaccines encoding internal antigens of influenza virus A/PR/8 (H1N1) on subsequent challenge with other subtypes, including H5N1 viruses from the 1997 outbreak in Hong Kong. Results vary with the virulence of the challenge virus, but protection is observed in some cases.